Isobutyl methacrylate

Endpoint summary

Additional information

Data availability

For i-BMA gene mutation data in bacteria and chromosome mutation data in vivo are available. For mammalian gene mutation data are inferred by category read-across from the smaller and larger structural analogues MMA and 2-EHMA.

In vitro assays

Bacterial gene mutation assay

The potential of isobutyl methacrylate to induce reverse mutation inSalmonella typhimurium(strains: TA98, TA100, TA1535, TA1537) was evaluated according to a protocol comparable to the OECD guidelines 471 (Zeiger et al., 1987). i-BMA was tested in single experiment, with and without a metabolic activation system, according to the preincubation method (20 min at 37 °C). Concentrations of i-BMA (0, 100, 333, 1000, 3333, and 10000 µg/plate.), overnight culture ofS. typhimurium(0.05-0.10 ml) and S-9 mix or buffer were incubated without shaking for 20 minutes. The top agar was added and the contents of the tubes were mixed and poured onto the surfaces of Petri dishes. His+ (histidine dependent) colonies arising on plates were machine-counted after two days incubation at 37 °C. Testing was without metabolic activation, with 10% rat liver S-9, or with 10% hamster liver S-9. The positive control chemicals induced a significant increase of the revertant frequency in all tester strains, either with or without metabolic activation. i-BMA was negative, in the presence and absence of metabolic activation, in all tester strains.This result is consistent with the close structural isomer n-BMA (Nakajima, 1998) and the results of other esters of the category in bacterial mutation tests.

Chromosomal aberration assay in mammalian cells

No in vitro data are available for i-BMA, but the structural isomer n-BMA was tested in anin vitrochromosomal aberration test in Chinese hamster lung fibroblasts (CHL; Nakajima et al., 1998b). The study was performed following the OECD 473 guideline and GLP. The concentrations were set based on the results of previously conducted cell growth inhibition tests. The test was performed at concentrations of 178, 355, 710 and 1420 µg n-BMA/ml (≈10mM) with treatments of 6, 24 and 48 hours (continuous treatment method) without metabolic activation and at concentrations of 355, 710 and 1420 µg n-BMA/ml for 6 hours (short term treatment) with metabolic activation.

Both the positive control substances mitomycin C (MMC) in the continuous treatment method and cyclophosphamide (CP) in the short term treatment method induced a high frequency of chromosomal structural aberrations. n-BMA did not induce a higher frequency of chromosomal aberration for both the continuous treatment method and short term treatment method.

In addition, in vivo chromosome mutation data are available for i-BMA (see below).

Gene mutation assay in mammalian cells

As there are no mammalian gene mutation data for i-BMA, data are used from the other members of the category: methyl methacrylate (CAS 80-62-6); ethyl methacrylate (CAS 97-63-2) and 2-ethyl-hexyl methacrylate (CAS 688-84-6)

Methyl methacrylate (CAS 80-62-6)

For gene mutations in mammalian cells data are used from MMA by read-across. Three mouse lymphoma assays were described for methyl methacrylate. According to Myhr et al. (1990) methyl methacrylate was weakly positive with and without S-9 mix. With S-9 mix a dose-dependent increase in mutation frequencies was obtained for doses ranging from 250 nl/ml (doubling of control level, 72% relative total growth) to 1,500 nl/ml (more than 3-fold the control level, relative total growth 25%). Without S-9 mix the substance was positive in 1 out of 2 experiments for doses ranging from 500 to 1,000 nl/ml; 1,500 nl/ml led to total toxicity. In a second experiment a weak positive response was obtained at 1,500 nl/ml. Clear and reproducible increases in mutation frequencies were bound to high toxicity below 20% relative total growth. Cifone (1985) reported on a mouse lymphoma assay which was weakly positive in presence and negative in absence of S-9 mix. Without S-9 mix doses up to 100 nl/ml were tested, higher doses led to total toxicity. With S-9 mix methyl methacrylate was positive in the dose range 100 nl/ml to 250 nl/ml, however, clear effects were observed only at doses with high toxicity below 20% relative growth. In a third mouse lymphoma assay which was only run without S-9 mix, weak effects were obtained for doses producing high toxicity (Moore et al., 1988). According to the authors, 2,000 µg/ml was positive in both experiments (92 and 98 mutants per 106 survivors vs. 54 and 68 in the negative controls), relative survival was approximately 20% and 30%; in one experiment the highest dose of 2,499 µg/ml induced 143 mutants at 10% relative survival; in the second experiment the highest dose of 3,100 µg/ml induced 220 mutants with 11% relative survival. The vast majority of induced colonies were small ones (indicating that the genetic effect was derived from clastogenicity and not from gene mutations).

In several in vitro tests MMA induced an increase in gene mutations – associated with the induction of chromosomal aberrations in the same dose range. This potential seems to be limited to high doses with strong toxic effects.

2-Ethyl-hexyl methacrylate (CAS 688-84-6)

The potential of 2-Ethylhexyl methacrylate to induce gene mutations at the HPRT locus in V79 tells of the Chinese hamster was investigated in an OECD guideline 476 and GLP study (Harlan, 2009). The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment period of 24 hours in the absence and 4 hours in the presence of metabolic activation. The maximum dose of the pre-test was 2000 µg/mL corresponding to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxic effects and was 0.1 - 16.0 (1^stexperiment) and 3.8 – 60.0 (2^ndexperiment) µg/ml without S9 and 62.5 – 2000 µg/ml with S9 (1^stand 2^ndexperiments). No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiments up to the maximum concentration. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies and thus showed the sensitivity of the test system and the activity of the S9 mix. In conclusion, 2-ethylhexyl methacrylate did not induce gene mutations at the HPRT locus in V79 cells.

In vivo assay

The ability of i-BMA to cause chromosomal damage in vivo was investigated in a micronucleus OECD 474 test (RCC. 1989). Dose-levels for treatment were selected on the basis of a preliminary toxicity test. Male and female NMRI mice were dosed once intraperitoneally with vehicle only, CMC 1% in water and 5000 mg/kg bw i-BMA and the positive control Cyclophosphamide. Six animals per sex from each group were sacrificed at the 24 hour sampling time. Further sampling times were at 48 and 72 hours. Following treatment with i-BMA, no statistically significant increase in the incidence of micronucleated PCE's over the control value was observed with 5000 mg/kg at any sampling time. Increases in the ratio of mature to polychromatic erythrocytes, compared to the vehicle control, most pronounced at the 72 h sampling time, were seen in both male and female animals from the substance dose group and the positive control, indicating that the test substance exerted a toxic effect on the bone marrow cells. Following treatment with the positive control Cyclophosphamide, statistically significant increases in the incidence of micronucleated PCE's over the control values were seen in the positive control group indicating the correct functioning of the test system. It is concluded that i-BMA administered orally by gavage at a dose-levels of 5000 mg/kg bodyweight to both male and female animals, does not induce micronuclei in the polychromatic erythrocytes of treated mice, under the reported experimental conditions.

Besides the negative micronucleus test, further support for the absence of genotoxic potential can be gained by read-across from MMA, referring to a dominant lethal test in CD-1 mice (Anderson and Hodge, 1976). In this study groups of 20 male CD-1 mice were exposed via inhalation to MMA at 100, 1000, or 9000 ppm (416, 4160 and 37440 mg/m³) for 6 h/day for 5 days. Each male was subsequently mated with 2 different unexposed female mice weekly over a period of 8 weeks. MMA did not induce dominant lethal mutations as indicated by no adverse effect on total implants and early or late post-implantation death in the offspring of treated males compared to controls.

Metabolites

It has been established that the lower alkyl methacrylates have a common mode of chemical reactivity via the C=C double bond and Michael addition and as such this lends them the potential to be chemically reactive towards macromolecules such as protein and DNA though a mechanism of electrophilic attack. It has also been established that these esters are subject to hydrolysis by ubiquitous carboxylesterases. The resultant acid and alcohol metabolites are non genotoxic. Intracellular generation of MAA is a potential mechanism through which intracellular pH could be reduced and this has been shown to lead to clastogenic effects in in vitro test systems, It is hypothesised, however, that these findings in vitro at high test concentrations are not relevant in vivo since systemic acidosis causes metabolic imbalance and is a lethal effect so that such high intracellular levels would not be achieved in vivo.

MAA:

EU ESR on MAA concluded: “Methacrylic acid is negative in a bacterial gene mutation test. Further testing on methacrylic acid is lacking. However, taking into consideration the data on the structurally related substance methyl methacrylate - which indicate that this substance does not express a genotoxic potential in vivo - there is no need for further testing.” Consistent with this assessment the missing endpoints are satisfied by cross-reading the key studies from MMA.

Alcohols:

None of the lower alkyl alcohol metabolites are regarded as mutagenic, including i-butanol.

Summary                                  

Overall, i-BMA is negative in bacterial gene mutation tests and from the category data there is data showing that it is expected to be negative in mammalian cells as well. i-BMA as well as the structural n-BMA did not induce chromosome aberrations in vitro or in vivo and this, also, is supported by the rest of the data in the methacrylates category. The primary metabolites methacrylic acid and i-butanol are non-mutagenic.

In conclusion, i-BMA is regarded as non-genotoxic.

 

Short description of key information:
Gene mutation in bacteria
Negative S. typhimurium TA 1535, TA 1537, TA 98, and TA 100 with and without metabolic activation (OECD 471) (Zeiger et al. 1987)
Gene mutation in mammalian cells
Negative HPRT test in V79 cells (by read-across from 2-ethylhexyl methacrylate), OECD 476, (Harlan, 2009)
and also the in vivo studies:
Cytogenicity in mammalian cells
Negative Chromosomal aberration test in CHL cells, with and without metabolic activation, OECD 473, (Nakajima, 1998b) read-across from n-BMA
In vivo
Negative Micronucleus assay, mouse bone marrow, OECD 474 (RCC, 1989)
Negative Dominant lethal assay in the mouse (by read-across from methyl methacrylate) (Anderson and Hodge, 1996)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to the available data and the CLP criteria for classification as germ cell mutagens, no classification is warranted for isobutyl methacrylate